Preparation and immunological properties of a nanovaccine against Pseudomonas aeruginosa based on gold nanoparticles and detoxified lipopolysaccharide

Objective(s): Pseudomonas aeruginosa is one of the most important bacterial pathogens in immunocompromised patients, and the lipopolysaccharide (LPS) of this organism is a key factor in virulence and both innate and acquired host responses to infection. In this study, we prepared a nanoconjugate vaccine composed of P. aeruginosa detoxified lipopolysaccharide (D-LPS) and gold nanoparticles (Au NPs) and evaluated its potential as a vaccine candidate against P. aeruginosa infections. Materials and Methods: LPS from P. aeruginosa strain PAO1 was extracted by the hot phenol method with some modifications and then detoxified. Au NPs were synthesized by the reduction of hydrochloroauric acid trihydrate by sodium borohydride and then coupled to D-LPS via electrostatic interaction. Mice were subcutaneously injected in the tail base with 20 µg of D-LPS, D-LPS-Au NPs, Au NPs, and PBS. IgG titers were evaluated by ELISA and whole-cell ELISA methods. The immunized and control group mice were challenged with a 2×LD50 (7.5×107 CFU) of P. aeruginosa strain PAO1.  Results: Mice vaccinated with D-LPS and D-LPS-Au NPs elicited a significant amount of IgG antibodies. Nanoconjugated LPS generated a significantly higher antibody titer compared with D-LPS alone. Also, immunization of mice with D-LPS-Au NPs increased survival times against challenge with 7.5×107 CFU (2×LD50) of P. aeruginosa strain PAO1.  Conclusion: Our results showed that the suggested vaccine composed of P. aeruginosa D-LPS and Au NPs had a significant potential to protect against P. aeruginosa infections.


Introduction
Pseudomonas aeruginosa is a gram-negative opportunistic pathogen that poses a major threat to immunocompromised individuals such as patients with cancer, burn wounds, or cystic fibrosis (1). The success of P. aeruginosa as a pathogen is due to its intrinsic and acquired antibiotic resistance mechanisms, infection in individuals with underlying diseases, ability to establish robust biofilms, and the production of numerous toxic products, all contribute to the difficulty in treating such life-threatening infections (2, 3). As mentioned, this bacterium has the ability to easily acquire antimicrobial resistance. The emergence of multiple drug-resistant strains has become a major clinical problem due to limited treatment options (4). Therefore, immunoprophylaxis and immunotherapy may be effective methods for controlling P. aeruginosa infections and due to this, development of a vaccine has become a necessity (5). A primary approach to developing a vaccine against this organism has involved searching for P. aeruginosa virulence factors that generate protective antibodies (6).
An important virulence factor of P. aeruginosa is the LPS located in the outer portion of the outer membrane which promotes infection by interference with the host immune response (1,7). LPS has been implicated as a critical virulence factor or protective antigen or both when examined in experimental animal models. Anti-LPS antibody has been shown to play a key role in protection against P. aeruginosa infections (8). Therefore, LPS is capable of acting as a target for effective immunity against P. aeruginosa infections (9).
Recently, Au NPs have received attention due to their potential use over traditional vaccine platforms. The location of Au NPs in lymphoid tissues and cells, their capacity of coupling to a variety of biomolecules, their stability, ease of their synthesis, their unique physical properties (size and shape-dependent), and their safety (essential for the development and synthesis of vaccines) provide a multifaceted focus for the design of this new vaccine platform (10,11). Furthermore, some in vitro and in vivo studies have revealed that various immune cells, including macrophages, dendritic cells, and lymphocytes, are stimulated by Au NPs leading to the production of proinflammatory cytokines (interleukin 1 β and tumor necrosis factor α) and T helper 1 cytokines (interferon γ and interleukin 2). Thus, the Au NPs can serve as both an antigen carrier and an adjuvant in immunization (12,13).

LPS characterization
The purified LPS was evaluated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with Coomassie blue staining. Analytical SDS-PAGE was carried out based on the method of Laemmli. Extracted LPS was dissolved in Laemmli sample buffer (Bio-Rad Laboratories, Inc., Hercules, CA, USA) and incubated at 95 °C for 5 min. Then, the sample was loaded in 4%-12% polyacrylamide gel (Mini-PROTEANVR TGX TM precast gels, Bio-Rad Laboratories, Inc.). Gel electrophoresis was performed at 60 mV for 3.5 hr (until the bromophenol blue reached the bottom of the gel). The gels were stained with the Coomassie brilliant blue R-250 staining solution (Bio-Rad Laboratories, Inc.) for 2 hr and destained overnight with the Coomassie brilliant blue R-250 destaining solution (Bio-Rad Laboratories, Inc.) (17). The protein content in the purified LPS was determined using the Bradford assay. To perform a Bradford assay, the purified LPS was added to microcentrifuge tubes and brought to a volume of 800 μl with water. Next, 200 μl of 5X Bradford reagent (Bio-Rad laboratories catalog number 500-0006) was added to the sample to bring it to a volume of 1 ml. The sample was then analyzed in a Beckman spectrophotometer to determine their absorbance at 595 nm. A standard curve was prepared with bovine serum albumin (BSA) as a control for all experiments. To do this, 2.5, 5, 7.5, 10, 12.5, 15, 17.5, and 20 μl of BSA at a concentration of 1 mg/ml was added to 800 μl of water and then used in the assay (18). LPS was analyzed for nucleic acid by measuring A 260 . The UV spectrophotometer, Nanodrop-ND1000 (Thermo Fisher Scientific, Waltham, MA), was used to measure DNA concentration, wherein 1 optical density at 260 nm is equivalent to 50 ng/μl of dsDNA (19).

LPS detoxification
For LPS detoxification, the LPS pellet was dissolved in NaOH to a final concentration of 0.2 N and heated for 2 hr at 100 °C. After cooling on ice, the pH was adjusted to 7 with 1 M HCl. LPS was dialyzed extensively against distilled water for 2 days at 4 °C with six changes of distilled water. Detoxified LPS (D-LPS) was harvested by cold absolute ethanol precipitation and pooled after centrifugation at 4000 × g for 45 min at 4 °C (14,16).

Pyrogenic test of D-LPS
The toxicity of D-LPS was assayed by the Limulus amebocyte lysate (LAL) test (Lonza, Switzerland) and expressed in endotoxin units related to the U.S. standard. For pyrogenicity studies, New Zealand White rabbits (2 to 2.5 kg, 3 in each group) were administered graded doses of D-LPS intravenously. Rectal temperatures were measured with indwelling rectal thermostats and recorded every 15 min for 1 hr before the challenge and every 15 min for 3 hr after the challenge. A negative response was less than a 0.5 °C rise in temperature at any time point (20).

Au NPs preparation and characterization
Hydrochloroauric acid trihydrate (HAuCl 4 3H 2 O) was purchased from Sigma-Aldrich Shanghai Trading Co Ltd, Shanghai, China. Au NPs were prepared with a phasetransferred process by the self-reduction method. Briefly, to a solution of HAuCl 4 in deionized water was added trisodium citrate (Sigma, USA) under vigorous stirring. HAuCl 4 was phase-transferred into the organic phase using sodium borohydride (NaBH 4 ) (Sigma, USA). Dropwise addition of a freshly prepared aqueous solution of reduction agent (NaBH 4 ) caused an instant color change. The resulting mixture was stirred at room temperature for an additional 5 hr. A wine-red organic phase containing Au NPs and a colorless aqueous phase were obtained. Therefore, the formation of gold nanoparticles was indicated by a visual color change from light yellow to wine red (21, 22).
The particle size distribution of Au NPs was analyzed using transmission electron microscopy (TEM) micrographs (Philips, em208s, USA) at an accelerating voltage of 100 kV (17,23).
Dynamic light scattering (DLS) was used to determine the mean size and size distribution of Au NPs. Measurements were made with a Malvern ZEN3600 (Malvern, ZEN3600, England). Zeta potential (Z-potential) (surface charge) measurements were used to determine the stability of a colloidal suspension of electrostatically stabilized Au NPs. Conversely, DLS allows the determination of the hydrodynamic diameters of colloidal particles and conjugates, i.e., the diameter of the sphere with the same Brownian motion as the particle under analysis (17,24).

D-LPS-Au NPs conjugate preparation
Hydrogen chloride-Potassium chloride (HCl-KCl) buffer (0.02 M, pH 2) was added to the Au surface. Then D-LPS was added to the above mixture under vigorous stirring and the resulting mixture was stirred for an additional 3 hr. The surface was washed with HCL-KCl buffer. The negatively charged backbones of D-LPS adsorbed on the positively charged Au NPs through electrostatic interaction and could potentially lead to the aggregation of (+) Au NPs (25,26).

Evaluation of nanoconjugate
Chemical analysis of nanoconjugate The nanoconjugate was chemically analyzed for protein level by the Bradford method and carbohydrate level by the phenol-sulfuric acid method (8,27). To perform the phenol-sulfuric acid method, to standard carbohydrate solutions (glucose at concentrations of 10-100 μg/ml) and sample, 200 μl of 5% phenol reagent and then 1 ml of concentrated sulfuric acid were added. After 10 min, the tubes were shaken vigorously for 30 min. Then, the absorbance of the tubes at 490 nm was read and the total carbohydrate concentration in the unknown sample was obtained using a standard curve (28).

Pyrogenic test of nanoconjugate
Rabbit pyrogen test for D-LPS-Au NPs was performed as previously described for D-LPS (20). The toxicity of the prepared nanoconjugate vaccine was evaluated in 5 groups of 6-8 week old female BALB/c mice (5 mice per group). Various doses of D-LPS-Au NPs (100, 150, 200, 250, and 300 μg per mouse) were subcutaneously injected in the tail base of mice.
Mice were monitored for any sign of illness or weightloss for 7 days after the challenge (29).

Physicochemical characterization of nanoconjugate
The average diameter of Au NPs after conjugation onto D-LPS was determined from the TEM micrographs using a Philips em208s electron microscope at an accelerating voltage of 100 kV (17,30).
DLS analysis (Malvern, ZEN3600, England) was used to determine the mean size and size distribution and check on the Z-potential of Au NPs after conjugation onto D-LPS (17,24).
Energy-dispersive X-ray spectroscopy (EDS) was used to analyze the elemental compositions of synthesized particles (31). EDS measurement was performed on a Compact Detector Unit (SAMX Detector, MIRA III, France) incorporated into a FESEM (TESCAN, Czech Republic). The EDS spectrum was obtained at an acceleration voltage of 20 kV. Mapping was completed using pseudo-color to represent the spatial distribution of energy emission from the Au element present in the sample (32).
The characterization of the chemical groups present in NPs obtained by Fourier transform infrared (FTIR). FTIR spectra over the wavelength range of 4000-400 cm -1 were recorded for D-LPS and D-LPS-Au NPs using an FTIR spectrometer (30,33). Samples were mixed with propidium iodide (PI)/water solution for a weight ratio of sample/dye of 2.6. The solution was allowed to dry to a purple powder. Samples were also dried to a thin gold film. PI powder was used as received. FTIR was performed on a Thermo AVATAR FTIR "Smart Orbit" (Thermo, AVATAR, USA). A background scan was performed prior to sample scanning. All scans were performed with 1024 scans at 4 cm -1 resolution (34).

Immunization of mice
The 6-8 weeks old female BALB/c mice were obtained from the Laboratory Animal Production Department at Pasteur Institute of Iran (Research & Production Complex, Alborz). Animals care, feeding, and all experiments were performed based on institutional ethical guidelines and international protocols (35).
Mice were divided into 4 experimental groups (each group included 7 mice) and subcutaneously injected in the tail base with 20 µg of D-LPS, D-LPS-Au NPs, Au NPs (test groups), and 300 μl PBS (control group) on days 0 (with complete Freund's adjuvant (Sigma, USA)), 14, 28, and 42 (with incomplete Freund's adjuvant (Sigma, USA)). One week after the last injection, the mice were bled from the orbital sinus, and sera were collected and stored at -20 °C until analysis (36).

Enzyme-linked immunosorbent assay (ELISA)
LPS-specific total IgG levels in sera of different mouse groups were determined by enzyme-linked immunosorbent assay (ELISA). Briefly, D-LPS, D-LPS-Au NPs, and Au NPs were diluted to 5 μg/ml with 0.1 mol/l carbonate/bicarbonate buffer (pH 9.6) and divided into 96-well flat-bottom microtiter plates (Nunc MaxiSorp, Sigma, USA) using 100 μl per well.
After 45 min incubation at 37 °C, plates were washed twice with PBS containing 0.05% Tween 20 (PBS-T). 250 μl of blocking buffer (2% BSA in PBS-T) was added to each well to block non-specific binding sites and plates were incubated for 1 hr at 37 °C. Then, the plates were washed twice with PBS-T. 100 µl of diluted mouse sera (1:100 dilution) was added to each well and incubated for 45 min at 37 °C. After washing the plates, 100 µl of diluted horseradish peroxidase-conjugated goat antimouse IgG (Abcam, USA) (1:5000 dilution) was added to each well and then plates were incubated for 45 min at 37 °C. After incubation and wash steps, 100 µl of 3, 3′, 5, 5′-tetramethylbenzidine (Sigma, USA) was added to the wells. After 15 min incubation in the dark, reactions were stopped using 1 mol/L H 2 SO 4 and the optical density (OD) was measured using an automated ELISA reader at 450 nm. ELISA test was performed in triplicate for all samples (37,38).

Whole-cell ELISA
A single colony of P. aeruginosa strain PAO1 was inoculated into LB broth medium and grown overnight at 37 °C with vigorous shaking. After incubation, all bacterial cells were collected, washed twice with PBS, and resuspended in PBS at a final concentration of 10 8 CFU/ml. The bacterial suspension (100 µl/well) was coated into ELISA microtiter plates, incubated for 1 hr at 37 °C, and washed twice with PBS-T. The blocking and addition of other reagents were performed as mentioned above for ELISA (39).

Opsonophagocytosis assay
The ability of vaccinated antisera to promote the uptake of P. aeruginosa by murine peritoneal macrophages was determined by the visual phagocytosis assay. For preparation of peritoneal macrophages, mice were injected intraperitoneally with 0.5 ml sodium thioglycollate. After 4 days, peritoneal exudate cells were harvested by washing the peritoneal cavity with 10 ml cold RPMI-1640 medium and aspirating the exudate with a syringe. The cells were washed twice with cold PBS and suspended in RPMI-1640 medium with 10% fetal calf serum (FCS), and finally, cell viability was evaluated by the dye exclusion method (40).
A mucoid colony of P. aeruginosa strain PAO1 was grown to mid-log phase on LB broth medium at 37 °C with shaking, washed twice with cold PBS, and suspended in PBS at a concentration of 10 6 CFU/ml. Opsonization was performed by tumbling 100 μl of bacteria (10 6 CFU/ml) for 30 min at 37 °C in 100 μl of heat-inactivated sera obtained from various vaccinated and control groups. After washing twice with PBS, 100 μl of 10 6 macrophages/ml and 10% fresh infant rabbit serum as a complement source were added to the mixture and incubated for 90 min at 37 °C. The control samples consisted of tubes from which macrophage, complement, or serum was omitted. After 90 min of incubation at 37 °C, 10 μl samples were removed and diluted in PBS. Finally, the samples were plated and incubated for 18 hr at 37 °C for bacterial enumeration. The opsonophagocytosis activity of the sera was determined as follows (27,29): Opsonic activity= [1-(CFU of immune serum at 90 min/ CFU of preimmune serum at 90 min)]×100

Challenge study
The 50% lethal dose (LD 50 ) of P. aeruginosa strain PAO1 was determined in 5 female BALB/c mice groups (7 mice per group, 6-8 weeks old) in order to estimate the infecting inoculum for in vivo protection test. Mice were intraperitoneally injected with 2.5×10 7 , 5×10 7 , 7.5×10 7 , 1×10 8 , and 12.5×10 8 CFU of P. aeruginosa strain PAO1 suspended in sterile PBS. Mice were observed for 10 days, mortality was recorded, and then LD 50 was determined according to the Reed and Muench method. For the bacterial challenge, 14 days after the last vaccination, the test and control group mice were intraperitoneally challenged with 7.5×10 7 CFU (2×LD 50 ) of P. aeruginosa strain PAO1 in sterile PBS. The survival rate was daily recorded in the challenged mice for 10 days. They were observed every 6 hr for the first 2 days and twice a day thereafter and mortality was recorded (29,41).

Statistical analysis
SPSS version 22.0 was used for data analysis. Differences in the mean ELISA absorbance of each group with the mean of the control group were compared by Student's t test. Values of P≤0.05 were considered to be significant. The ratio of live mice/total mice (survival percent) was used for the survival study (27,29).

Evaluation of D-LPS
Coomassie blue staining of LPS from P. aeruginosa strain PAO1 by SDS-PAGE revealed a progressive ladderlike pattern of bands up the gel (Figure 1). For members of the family Enterobacteriaceae and P. aeruginosa strains, these bands have been reported to represent LPS molecules containing increasing lengths of O antigen (42). The protein and nucleic acid amounts of the purified LPS were 0.5 μg/ml, and 0.5 μg/ml, respectively. The amount of endotoxin in LPS, estimated by the LAL test, was 0.125 endotoxin units per ml. There were not pyrogenic in rabbit thermal tests.

Evaluation of nanoconjugate
The protein and carbohydrate contents of D-LPS-Au NPs were 0.5 μg/ml, and 0.4 μg/ml, respectively. D-LPS-Au NPs conjugate was non-pyrogenic and evoked <0.5 °C increase in body temperature of each rabbit after 24 hr. Toxicity test of D-LPS-Au NPs showed that none of the mice injected with various doses of nanoconjugate (between 100 and 300 μg) died after 7 days.
Au NPs before and after conjugation onto D-LPS were imaged using TEM (Figure 2). Au NPs after conjugation onto D-LPS had a mean diameter of 14 nm ( Figure 2B). D-LPS binding to Au NPs was assessed by DLS and Z-potential in comparison with Au NPs (Figure 3). As shown in Figures 3A and B, Au NPs showed a size increase via electrostatic interaction to D-LPS ( Figures  3A and B). In addition to increased Au NPs diameter after conjugation, a decrease in Z-potential indicates D-LPS binding to the Au NPs ( Figures 3C and D).
EDS mapping analysis demonstrates that Au NPs were loaded as a homogeneous dispersion in D-LPSAu-NPs ( Figure 4A). The EDS spectrum of D-LPS-Au NPs showed Au signals, which confirmed the presence of elemental Au in the sample ( Figure 4B). FTIR spectroscopy was used for structural analysis of the conjugated NPs and conjugation bonds. Figure 5 shows FTIR spectra of D-LPS (spectrum A) and D-LPS-Au NPs (spectrum B) in the spectral region 4000-400 cm −1 . As seen in Figure 5, peaks spreading in the areas of 1120-1000 cm -1 and 1760-1710 cm -1 are related to the formation of P=O and C=O bonds, respectively, and these spectra confirmed the binding of Au NPs to D-LPS. The

ELISA of immune responses
One week after the last injection, the levels of IgG against D-LPS, D-LPS-Au NPs, and Au NPs were measured by indirect ELISA and whole-cell ELISA methods. The antibody levels were compared with each other. Comparisons of experimental groups were carried out at 1:100 antibody dilution.
In indirect ELISA, both D-LPS and D-LPS-Au NPs elicited a statistically significant IgG antibody response but neither Au NPs nor PBS evoked antibody (P≤0.05) ( Table 2). As shown in Table 2, D-LPS-Au NPs displayed higher titer in the IgG antibody than D-LPS (P≤0.05).   Similar results were obtained using the whole bacterial cell as a coating antigen in ELISA, where the highest titer was observed in mice immunized with D-LPS-Au NPs (P≤0.05) ( Table 2). D-LPS also indicated a Lower antibody titer than D-LPS-Au NPs. Additionally, neither Au NPs nor PBS elicited antibody (P≤0.05).

Opsonophagocytosis assay
The ability of antibodies induced by D-LPS, D-LPS-Au NPs, Au NPs, and PBS to promote the uptake and killing of mucoid P. aeruginosa strain PAO1 is shown in Figure 6. Immunization with D-LPS-Au NPs evoked a substantial opsonic antibody response against P. aeruginosa strain PAO1 in comparison with other groups. There was no phagocytic killing when antibody, complement, or macrophages were omitted.

Challenge study
The LD 50 for P. aeruginosa PAO1 was determined according to the Reed and Muench method and was found to be 3.75×10 7 CFU and 7.5×10 7 CFU was selected as 2 × LD 50 . Mice immunized with D-LPS and D-LPS-Au NPs showed significant protection against intraperitoneal challenge with 7.5×10 7 CFU (2×LD 50 ) of P. aeruginosa strain PAO1. This challenge dose killed 6/7 of mice that were injected with Au NPs and PBS, and 1/7 of mice immunized with D-LPS and D-LPS-Au NPs. Comparison of mice mortality times in immunized groups with D-LPS and D-LPS-Au NPs showed that immunization with D-LPSAu NPs increased the survival time of mice against intraperitoneal challenge with an approximate 7.5×10 7 CFU (2×LD 50 ) of P. aeruginosa strain PAO1 (Figure 7). These results are in good correlation with the IgG titers and also with opsonophagocytosis assay of the sera.

Discussion
P. aeruginosa is an important cause of nosocomial infections leading to significant morbidity and mortality (29). Effective treatment and control of P. aeruginosa infections remain a persistent problem, because of the natural resistance of the bacterium and its remarkable ability to acquire resistance to multiple antimicrobial agents (43). Due to antibiotic resistance, the development of a vaccine against P. aeruginosa is an appropriate and challenging strategy to pursue (44).
The LPS of P. aeruginosa is a key factor in virulence and both innate and acquired host responses to infection. Previous studies established that the conserved epitope of LPS in this organism had efficacy in inducing cytokine responses. In addition, LPS can bind to Toll-like receptor 4 (TLR4) on the surface of immune cells and stimulate inflammatory immune responses (9,45). Despite extensive studies, LPS-based vaccines are not successful owing to their low immunogenicity (43). To overcome this problem, we conjugated D-LPS onto Au NPs to produce an effective nanovaccine against P. aeruginosa infections. For the first time, Au NPs were used as carriers of haptens for antibody preparation in 1986 (46). Since then, many studies have been focused on Au NPs as an antigen carrier and adjuvant in immunization for the development of effective vaccines (47)(48)(49)(50).
In this study, the LPS from P. aeruginosa strain PAO1 was investigated for a nanovaccine preparation, we extracted the LPS by the modified hot phenol with TCA procedure. A study showed that among extraction methods of LPS (extraction by butanol with enzymatic digestion, hot phenol extraction with enzymatic digestion, and modified hot phenol with TCA procedure), the yield of LPS extraction in the modified hot phenol with TCA procedure is 1.5 times the yields of the other two methods and accordingly this procedure is the best method for LPS production (15).
The Au NPs were prepared by using the Natan method. To produce Au NPs, Brown et al. introduced a method using citrate as a stabilized agent and borohydride as a reducing agent. The Au NPs were obtained by adding the NaBH 4 /citrate mixture into the HAuCl 4 solution at room temperature. With this method, the size of Au NPs is tailored to 6 nm (51). Then we coupled the synthesized Au NPs to D-LPS via electrostatic interaction.
After vaccination of mice, specific IgG titers were determined by ELISA and whole-cell ELISA methods.  Abu-baker et al. used both ELISA and whole-cell ELISA methods to measure specific IgG anti-P. aeruginosa LPS antibody levels in collected sera from mice immunized with O-polysaccharide recombinant exoprotein A conjugate and similar results were obtained using both ELISA methods (39). Similarly, in this study, the wholecell ELISA was used to ensure that the LPS orientation did not change during the extraction procedure. Although we used two different coating antigens (whole bacteria and LPS), we obtained similar results with respect to the antibody titer. This indicates that the LPS isolation procedure did not change the original state of the molecules.
Our results show that D-LPS conjugated to Au NPs induced antibody responses which were superior to the responses induced by D-LPS alone. Since the coupling chemistry is selective for nucleophilic carbonyl groups in the LPS core region, this leaves the O-antigen and lipid A moieties exposed. Whilst LPS O-antigen provides epitopes for the development of antigenspecific antibodies, the lipid A region is a potent agonist for TLR4 and evokes a strong immune response from interaction with B memory cells. Additionally, the increased response from D-LPS-Au NPs may be due to the presentation of LPS in its coupled form and the high density of O-antigen epitopes localized on an Au NP surface compared with uncoupled DLPS alone (48). Other workers have reported the ability of Au NPs to enhance the ability of an antigen to evoke antibody responses compared with antigen given alone. Gregory et al. covalently coupled Au NPs with one of three different protein carriers TetHc (Hc fragment of tetanus toxin), Hcp1 (hemolysin co-regulated protein produced by both Burkholderia mallei and Burkholderia pseudomallei) and FliC (flagellin produced by B. pseudomallei)) followed by conjugation to LPS purified from a nonvirulent B. thailandensis strain. Mice were immunized three times intranasally. When they compared with LPS alone, the Au NPs-protein-LPS conjugates were found to generate significantly higher antibody titers (48). A similar approach was developed for vaccination against B. pseudomallei myeloidosis. Resaerchers immunized mice three times subcutaneously with Au NPs glycoconjugate vaccines composed of B. thailandensis LPS conjugated to the novel flagellar protein FlgL and a protein combination (FlgL, Hcp1, and hemagglutinin). Mice vaccinated with Au NPs glycoconjugate vaccines generated significantly higher antibody titers than did native antigens (52). Another study examined the effects of LPS adsorption on the NP surface on the formation of a biocorona in biological fluids and on the subsequent inflammation-inducing activity of NPs. Different Au NPs were exposed to Escherichia coli LPS under different conditions. LPS coated Au NPs, but not the LPS-free NPs, induced significant inflammatory responses in human monocytes in vitro (17). A study investigated the effect of intraperitoneal treatment using Au NPs on the inflammatory response and pulmonary oxidative stress in Wistar rats induced by LPS. The results showed that the LPS + Au NPs group exhibited a significant decrease in the levels of the pro-inflammatory cytokines, activities of antioxidant enzymes, oxidative stress parameters, and total leukocyte proliferation compared with the LPS group (53).
In the opsonophagocytic killing assay, D-LPS conjugated to Au NPs was superior to D-LPS alone in its ability to induce opsonic antibodies in mice. The results found here were consistent with the findings of Safari et al. They reported sera obtained from mice immunized with Au glyconanoparticles, bearing a synthetic tetrasaccharide epitope related to the Streptococcus pneumoniae type 14 capsular polysaccharide, were able to opsonize S. pneumonia type 14 bacteria (54).
Immunization with D-LPS and D-LPS-Au NPs afforded significant protection against challenge with P. aeruginosa strain PAO1 in comparison with Au NPs and control groups.
However, there was no significant difference between the survival of mice immunized with D-LPS or D-LPS-Au NPs, but the survival times of mice immunized with the D-LPS-Au NPs were longer than the survival times of mice immunized with D-LPS. These findings were consistent with the results reported by Gregory et al. Three weeks after the final immunization boost, they tested immunized mice with Au NPs-one of three different protein carriers (TetHc, Hcp1, and FliC)-B. thailandensis LPS conjugates for their protective capacity against 3.5 × LD 50 of B. mallei challenge and recorded the meantime to death. Gregory et al. indicated that there was no significant difference between the survival of immunized mice with LPS, Au NPs-LPS, or Au NPs-TetHc-LPS (48).

Conclusion
In general, our results showed that coupling P. aeruginosa D-LPS onto Au NPs markedly increased both the levels of IgG and opsonic antibodies and the survival times of immunized mice. Therefore, D-LPS-Au NPs can be used as a candidate vaccine against P. aeruginosa infections.